Journal of Invertebrate Pathology 105 (2010) 329–334 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip Hematodinium sp. infection of red Paralithodes camtschaticus and blue Paralithodes platypus king crabs from the Sea of Okhotsk, Russia ⇑ T.V. Ryazanova a, M.G. Eliseikina b, A.D. Kukhlevsky b, V.I. Kharlamenko b, a Kamchatka Research Institute of Fisheries and Oceanography, Petropavlovsk-Kamchatsky 683002, Russia b A.V. Zhirmunsky Institute of Marine Biology FEB RAS, Palchevsky str. 17, Vladivostok 690041, Russia article info abstract Article history: A disease caused by a parasitic dinoflagellate of the genus Hematodinium was identified in red, Paralithodes Received 8 December 2009 camtschaticus, and blue, Paralithodes platypus, king crabs from the north-east region of the Sea of Okhotsk, Accepted 29 July 2010 Russia, during annual stock surveys. No carapace color change was observed even in heavily infected crabs, Available online 5 August 2010 but diseased crabs possessed creamy-yellow hemolymph, which was visible through the arthrodial mem- branes of the abdomen and appendages. Several stages of the parasite’s life history, including trophonts, Keywords: plasmodia, sporonts and macrodinospores, were observed in tissues of infected king crabs. Numerous par- Hematodinium sp. asite cells were observed in the lumina of the myocardium, the gills, the connective tissue of antennal Paralithodes camtschaticus glands and the sinuses of nerve ganglia, eyestalks and gastrointestinal tract of king crabs with gross signs Paralithodes platypus Sea of Okhotsk of infection. Based on sequencing of the 18S rDNA, it appears that the Hematodinium sp. found in red and Infection blue king crabs is identical or closely related to Hematodinium sp. isolated from crabs of the genera Chion- Prevalence oecetes and Lithodes. Observed prevalences were 0.33% in sublegal male red king crabs, 0.18% in female red Parasitic dinoflagellate king crabs, 0.34% in sublegal male blue king crabs and 0.31% in female blue king crabs. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction crab ( Chionoecetes bairdi) hemolymph developed detectable infections; and hemolymph smears from southeast Alaska crabs, The red king crab, Paralithodes camtschaticus, followed by the P. camtschaticus, P. platypus, and Lithodes aequispina contained no blue king crab, Paralithodes platypus, are the most commercially dinoflagellate forms (Meyers et al., 1987). The first incident of valuable crab species in the Sea of Okhotsk, Russia. Combined an- Hematodinium sp. infection was observed on the West Kamchatka nual landings of these species total 50,000 metric tons and over re- shelf in 2002 in snow crabs, Chionoecetes opilio, and this disease cent decades, these landings have been stable when compared to was found in red and blue king crabs, P. platypus, from this area other regions (Otto and Jamieson, 2001). However, since 1999, four years later. the Sea of Okhotsk king crab stock has declined significantly and This report describes the results of gross or macroscopic exam- remains low (Dolgenkov and Koblikov, 2009). In addition to overf- ination as well as microscopic (light and transmission electron ishing, a number of other factors may impact crab population microscopy) observations of the infection in king crabs P. camtsch- structure, including diseases. In particular, infections caused by aticus and P. platypus, molecular identification of Hematodinium sp. the parasitic dinoflagellate Hematodinium sp., may significantly in king crabs, and it also includes information on the distribution of change the size and structure of important crab populations (Sten- infected crabs in the north-east region Sea of Okhotsk. tiford and Shields, 2005). Hematodinium sp. infections in various marine crustacean spe- 2. Materials and methods cies have been recorded in many areas of the world. Hematodini- um-associated diseases are generally fatal to the host (Messick Lithodid crabs, the red king crab P. camtschaticus and blue king and Shields, 2000), and its occurrence can reach 100% for some crab P. platypus, were used as material for the present research. The crustacean species (Messick, 1994). The majority of infections are survey took place in the northeastern area of the Sea of Okhotsk reported in brachyuran crabs (Stentiford and Shields, 2005). In during annual stock assessment surveys (Fig. 1). Crab trap and disease transmission studies, Meyers et al. (1987) reported that trawl surveys were used. The crab trap survey onboard the RV none of the lithodid king crabs inoculated with infected Tanner Ametist from August 25 to December 15 2006 was conducted from the north to the south. Traps were deployed in ‘fleets’ of 100 stan- ⇑ Corresponding author. dard Japanese conical traps baited with 1 kg of frozen herring. E-mail address: [email protected] (V.I. Kharlamenko). Eighty-eight fleets of traps were set between 64 and 322 m depth 0022-2011/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2010.07.009 330 T.V. Ryazanova et al. / Journal of Invertebrate Pathology 105 (2010) 329–334 drated in acetone and embedded in Epon-Araldite. Semithin and ultrathin sections were cut using a Leica EM UC6 ultramicrotome. Semithin sections were stained with methylene blue and examined using a Leica DM 4500 microscope. Ultrathin sections were stained with uranyl acetate and lead citrate and observed with a Zeiss Libra 120 transmission electron microscope. The standard method for total DNA isolation from the tissue of crabs was used (Sambrook et al., 1989). Hematodinium-specific primers Hemat-F-1487 and Hemat-R-1654 were used to detect Hematodinium (Gruebl et al., 2002). PCR was performed using a GenAMP 9600 thermal cycler system (Perkin Elmer). Amplification conditions for the PCR included an initial denaturation step (94 °C for 10 min) and 30 amplification cycles (denaturation, 15 s at 94 °C; annealing, 15 s at 56 °C; elongation, 30 s at 72 °C). Reaction products were checked for size and purity on 1% agarose gels. To confirm the identity of the parasites detected in king crabs, repre- sentative 196 bp PCR amplicons were used as templates for sequencing amplification using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Purified sequencing products were analyzed by electrophoresis on a 50 cm capillary array of an ABI Prism 3130 DNA sequencer. Sequences were assembled with SeqScape v2.5 software (Applied Biosystems). In addition, a long segment of 18S rDNA (1682 bp) from the parasite detected in the red king crab was sequenced. Direct sequence determination of each of the obtained amplicons yielded a 1464 bp sequence. The consensus sequences were compared with homologous Hematodi- nium 18S rDNA sequences available in GeneBank. Calculation of similarity values was conducted using the program MEGA version 4(Tamura et al., 2007). The density of infected crabs was estimated with the program Chartmaster version 3.1 using the kriging interpolation method. Fig. 1. Map of the study area with sampling stations. 3. Results at randomly selected locations in the range of latitudes from 58°510Nto57°10N and from 55°410Nto54°150N. The standard soak Overall, 11882 red king crabs and 4790 blue king crabs were time for traps was 36–48 h, but in some cases varied from 35 to subjected to gross examination. Gross signs of infection were 190 h depending on weather conditions. The 2007 trawl survey found in 14 red king crabs and nine blue king crabs. All infected was conducted onboard the RV Professor Kaganovsky from July 8 crabs were females or sublegal males (with the carapace width to August 1 beginning in the south and proceeding north. 146 from 75 to 102 mm) in the third intermolt stage (carapace and che- trawls from 51°050Nto57°420N were carried out within depth la are hard and cannot be depressed by thumb). No external range 14–200 m. The trawl sections were performed in a standard wounds were visible. None of the collected crabs changed color pattern at a speed of 3 knots for 30 min, using a DT 27.1 trawl. of their carapace, but infected crabs did possess a creamy-yellow All crabs were removed from the catch, sorted by species and hemolymph, which was visible through the arthrodial membranes sex. Crab size is reported as carapace width excluding spines. Crabs of abdomen and appendages (Fig. 2). Their dissection revealed that were divided into three groups – legal males (carapace width (cw) the hemolymph of suspect crabs appeared as a creamy-yellow fluid greater than or equal to 150 mm and 130 mm for red and blue mass surrounding the muscles of appendages and all internal or- kings crabs, respectively), sublegal (cw < 150 and 130 mm) for gans, filling the pericardial cavity and gill lamellae and stems. males and females, respectively. Intermolt state of the carapace The cooked meat of suspect crabs had a bitter and astringent taste. was assessed and assigned to one of six classes according to spe- Prevalence was 0.33%, 0.18%, 0.34% and 0.31% in sublegal male and cific criteria (Lysenko, 2001). In total, 3189 males and 1601 females legal female red king crabs, and legal female and sublegal male of the blue king crab, and 8587 males and 3295 females of the red blue king crabs, respectively. Gross evidence of infections was king crab were examined macroscopically. Randomly selected not found in legal male red or blue king crabs (Table 1). Although crabs with external signs of disease and additional individuals that the data are limited, prevalence of infection was different in trawl appeared healthy upon gross examination were dissected (700 and pot surveys. Infected crabs were found at depths ranging from males, 282 females of the red king crab, 230 males and 147 females to 30 to134 m. Although small in number, the majority of infected of the blue crab).